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MOTIVATION: Analysis of next-generation sequencing data often results in a list of genomic regions. These may include differentially methylated CpGs/regions, transcription factor binding sites, interacting chromatin regions, or GWAS-associated SNPs, among others. A common analysis step is to annotate such genomic regions to genomic annotations (promoters, exons, enhancers, etc.). Existing tools are limited by a lack of annotation sources and flexible options, the time it takes to annotate regions, an artificial one-to-one region-to-annotation mapping, a lack of visualization options to easily summarize data, or some combination thereof. RESULTS: We developed the annotatr Bioconductor package to flexibly and quickly summarize and plot annotations of genomic regions. The annotatr package reports all intersections of regions and annotations, giving a better understanding of the genomic context of the regions. A variety of graphics functions are implemented to easily plot numerical or categorical data associated with the regions across the annotations, and across annotation intersections, providing insight into how characteristics of the regions differ across the annotations. We demonstrate that annotatr is up to 27× faster than comparable R packages. Overall, annotatr enables a richer biological interpretation of experiments. AVAILABILITY AND IMPLEMENTATION: http://bioconductor.org/packages/annotatr/ and https://github.com/rcavalcante/annotatr. CONTACT: rcavalca@umich.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular/métodos , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Cromatina/metabolismo , Exones , Genómica/métodos , Polimorfismo de Nucleótido SimpleRESUMEN
MOTIVATION: Capabilities in the field of metabolomics have grown tremendously in recent years. Many existing resources contain the chemical properties and classifications of commonly identified metabolites. However, the annotation of small molecules (both endogenous and synthetic) to meaningful biological pathways and concepts still lags behind the analytical capabilities and the chemistry-based annotations. Furthermore, no tools are available to visually explore relationships and networks among functionally related groups of metabolites (biomedical concepts). Such a tool would provide the ability to establish testable hypotheses regarding links among metabolic pathways, cellular processes, phenotypes and diseases. RESULTS: Here we present ConceptMetab, an interactive web-based tool for mapping and exploring the relationships among 16 069 biologically defined metabolite sets developed from Gene Ontology, KEGG and Medical Subject Headings, using both KEGG and PubChem compound identifiers, and based on statistical tests for association. We demonstrate the utility of ConceptMetab with multiple scenarios, showing it can be used to identify known and potentially novel relationships among metabolic pathways, cellular processes, phenotypes and diseases, and provides an intuitive interface for linking compounds to their molecular functions and higher level biological effects. AVAILABILITY AND IMPLEMENTATION: http://conceptmetab.med.umich.edu CONTACTS: akarnovsky@umich.edu or sartorma@umich.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metabolómica , Programas Informáticos , Conjuntos de Datos como Asunto , Humanos , Redes y Vías Metabólicas , Estadística como Asunto , Vocabulario ControladoRESUMEN
MOTIVATION: Functional enrichment testing facilitates the interpretation of Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) data in terms of pathways and other biological contexts. Previous methods developed and used to test for key gene sets affected in ChIP-seq experiments treat peaks as points, and are based on the number of peaks associated with a gene or a binary score for each gene. These approaches work well for transcription factors, but histone modifications often occur over broad domains, and across multiple genes. RESULTS: To incorporate the unique properties of broad domains into functional enrichment testing, we developed Broad-Enrich, a method that uses the proportion of each gene's locus covered by a peak. We show that our method has a well-calibrated false-positive rate, performing well with ChIP-seq data having broad domains compared with alternative approaches. We illustrate Broad-Enrich with 55 ENCODE ChIP-seq datasets using different methods to define gene loci. Broad-Enrich can also be applied to other datasets consisting of broad genomic domains such as copy number variations. AVAILABILITY AND IMPLEMENTATION: http://broad-enrich.med.umich.edu for Web version and R package. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Inmunoprecipitación de Cromatina/métodos , Genómica/métodos , Histonas/metabolismo , Línea Celular , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Logísticos , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Background: DNA methylation plays a critical role in asthma development, but differences in DNA methylation among adults with varying asthma severity or asthma endotypes are less well-defined. Objective: To examine how DNA methylomic patterns differ among adults with asthma based on asthma severity and airway inflammation. Methods: Peripheral blood T cells from 35 adults with asthma in Beijing, China were serially collected over time (130 samples total) and analyzed for global DNA methylation using the Illumina MethylationEPIC Array. Differential methylation was compared among subjects with varying airway inflammation and severity, as measured by fraction of exhaled nitric oxide, forced expiratory volume in one second (FEV1), and Asthma Control Test (ACT) scores. Results: Significant differences in DNA methylation were noted among subjects with different degrees of airway inflammation and asthma severity. These differences in DNA methylation were annotated to genes that were enriched in pathways related to asthma or T cell function and included gene ontology categories related to MHC class II assembly, T cell activation, interleukin (IL)-1, and IL-12. Genes related to P450 drug metabolism, glutathione metabolism, and developmental pathways were also differentially methylated in comparisons between subjects with high vs low FEV1 and ACT. Notable genes that were differentially methylated based on asthma severity included RUNX3, several members of the HLA family, AGT, PTPRC, PTPRJ, and several genes downstream of the JAK2 and TNF signaling pathway. Conclusion: These findings demonstrate how adults with asthma of varying severity possess differences in peripheral blood T cell DNA methylation that contribute to the phenotype and severity of their overall disease.
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Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer (TNBC) with histological evidence of epithelial to mesenchymal transition (EMT) and aberrant differentiation. Inactivation of the tumor suppressor gene CCN6 (also known as WISP3) is a feature of mBrCAs, and mice with conditional inactivation of Ccn6 in mammary epithelium (Ccn6-KO) develop spindle mBrCAs with EMT. Elucidation of the precise mechanistic details of how CCN6 acts as a tumor suppressor in mBrCA could help identify improved treatment strategies. Here, we showed that CCN6 interacts with the Wnt receptor FZD8 and co-receptor LRP6 on mBrCA cells to antagonize Wnt-induced activation of ß-catenin/TCF-mediated transcription. The histone methyltransferase EZH2 was identified as a ß-catenin/TCF transcriptional target in Ccn6-KO mBrCA cells. Inhibiting Wnt/ß-catenin/TCF signaling in Ccn6-KO mBrCa cells led to reduced EZH2 expression, decreased histone H3 lysine 27 trimethylation, and deregulation of specific target genes. Pharmacological inhibition of EZH2 reduced growth and metastasis of Ccn6-KO mBrCA mammary tumors in vivo. Low CCN6 is significantly associated with activated ß-catenin and high EZH2 in human spindle mBrCAs compared to other subtypes. Collectively, these findings establish CCN6 as a key negative regulator of a ß-catenin/TCF-EZH2 axis and highlight inhibition of ß-catenin or EZH2 as a potential therapeutic approach for patients with spindle mBrCAs.
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BACKGROUND: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. OBJECTIVE: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. METHODS: Female mice were exposed to human relevant doses of either Pb (32 ppm) via drinking water or DEHP (5mg/kg-day) via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. RESULTS: The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n=13 and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types (n=55 and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. CONCLUSIONS: These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.
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Metilación de ADN , Impresión Genómica , Plomo , Hígado , Animales , Metilación de ADN/efectos de los fármacos , Ratones , Femenino , Hígado/efectos de los fármacos , Masculino , Plomo/toxicidad , Plomo/sangre , Impresión Genómica/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Encéfalo/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Exposición Materna , Ácidos Ftálicos/toxicidad , Embarazo , Efectos Tardíos de la Exposición Prenatal , Epigénesis Genética/efectos de los fármacosRESUMEN
BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are chemicals that are resistant to degradation and ubiquitous in our environments. PFAS may impact the developing epigenome, but current human evidence is limited to assessments of total DNA methylation. We assessed associations between first trimester PFAS exposures with newborn DNA methylation, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC). DNA methylation mediation of associations between PFAS and birth outcomes were explored in the Michigan Mother Infant Pairs cohort. Nine PFAS were measured in maternal first trimester blood. Seven were highly detected and included for analysis: PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnDA, and MeFOSAA. Bisulfite-converted cord blood DNA (n = 141) and oxidative-bisulfite-converted cord blood (n = 70) were assayed on Illumina MethylationEPIC BeadChips to measure total DNA methylation (5-mC + 5-hmC) and 5-mC/5-hmC. Correcting for multiple comparisons, beta regressions were used to assess associations between levels of PFAS and total methylation, 5-mC, or 5-hmC. Nonlinear mediation analyses were used to assess the epigenetic meditation effect between PFAS and birth outcomes. RESULTS: PFAS was significantly associated with total methylation (q < 0.05: PFHxS-12 sites; PFOS-19 sites; PFOA-2 sites; PFNA-3 sites; PFDA-4 sites). In 72 female infants and 69 male infants, there were sex-specific associations between five PFAS and DNA methylation. 5-mC and 5-hmC were each significantly associated with thousands of sites for PFHxS, PFOS, PFNA, PFDA, PFUnDA, and MeFOSAA (q < 0.05). Clusters of 5-mC and 5-hmC sites were significant mediators between PFNA and PFUnDA and decreased gestational age (q < 0.05). CONCLUSIONS: This study demonstrates the mediation role of specific types of DNA methylation on the relationship between PFAS exposure and birth outcomes. These results suggest that 5-mC and 5-hmC may be more sensitive to the developmental impacts of PFAS than total DNA methylation.
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Contaminantes Ambientales , Fluorocarburos , Embarazo , Recién Nacido , Humanos , Masculino , Lactante , Femenino , Madres , Metilación de ADN , MichiganRESUMEN
DNA methylation is a vital early step in carcinogenesis. Most findings of aberrant DNA methylation in head and neck squamous cell carcinomas (HNSCC) are array based with limited coverage and resolution, and mainly explored by human papillomavirus (HPV) status, ignoring the high heterogeneity of this disease. In this study, we performed whole-genome bisulfite sequencing on a well-studied HNSCC cohort (n = 36) and investigated the methylation changes between fine-scaled HNSCC subtypes in relation to genomic instability, repetitive elements, gene expression, and key carcinogenic pathways. The previously observed hypermethylation phenotype in HPV-positive (HPV+) tumors compared with HPV-negative tumors was robustly present in the immune-strong (IMU) HPV+ subtype but absent in the highly keratinized (KRT) HPV+ subtype. Methylation levels of IMU tumors were significantly higher in repetitive elements, and methylation showed a significant correlation with genomic stability, consistent with the IMU subtype having more genomic stability and better prognosis. Expression quantitative trait methylation (cis-eQTM) analysis revealed extensive functionally-relevant differences, and differential methylation pathway analysis recapitulated gene expression pathway differences between subtypes. Consistent with their characteristics, KRT and HPV-negative tumors had high regulatory potential for multiple regulators of keratinocyte differentiation, which positively correlated with an expression-based keratinization score. Together, our findings revealed distinct mechanisms of carcinogenesis between subtypes in HPV+ HNSCC and uncovered previously ignored epigenomic differences and clinical implications, illustrating the importance of fine-scale subtype analysis in cancer. Significance: This study revealed that the previously observed hypermethylation of HPV(+) HNSCC is due solely to the IMU subtype, illustrating the importance of fine-scale subtype analysis in such a heterogeneous disease. Particularly, IMU has significantly higher methylation of transposable elements, which can be tested as a prognosis biomarker in future translational studies.
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Neoplasias de Cabeza y Cuello , Infecciones por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Metilación de ADN/genética , Infecciones por Papillomavirus/complicaciones , Carcinogénesis , Inestabilidad Genómica , Virus del Papiloma Humano , Neoplasias de Cabeza y Cuello/genéticaRESUMEN
Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects. Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5-7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15. Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood. Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects.
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Background: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. Objective: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. Methods: Female mice were exposed to human relevant doses of either Pb (32ppm) via drinking water or DEHP (5 mg/kg-day) via chow for two weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and Chipenrich were used for genomic annotations and geneset enrichment tests of DMRs, respectively. Results: The cortex contained the majority of DMRs associated with Pb (69%) and DEHP (58%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n = 17 and 14 DMRs with Pb and DEHP exposure, respectively) and exposure types (n = 79 and 47 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, with 15 and 17 ICR-located DMRs across cortex, blood, and liver in each gene, respectively. The ICRs were also the location of DMRs replicated across target and surrogate tissues, suggesting epigenetic changes these regions may be potentially viable biomarkers. Conclusions: We observed Pb- and DEHP-specific DNAm changes in cortex, blood, and liver, and the greatest degree of overlap in DMR signatures was seen between exposures followed by sex and tissue type. DNAm at imprinted control regions was altered by both Pb and DEHP, highlighting the susceptibility of genomic imprinting to these exposures during the perinatal window of development.
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BACKGROUND: Revealing the gene targets of distal regulatory elements is challenging yet critical for interpreting regulome data. Experiment-derived enhancer-gene links are restricted to a small set of enhancers and/or cell types, while the accuracy of genome-wide approaches remains elusive due to the lack of a systematic evaluation. We combined multiple spatial and in silico approaches for defining enhancer locations and linking them to their target genes aggregated across >500 cell types, generating 1860 human genome-wide distal enhancer-to-target gene definitions (EnTDefs). To evaluate performance, we used gene set enrichment (GSE) testing on 87 independent ENCODE ChIP-seq datasets of 34 transcription factors (TFs) and assessed concordance of results with known TF Gene Ontology annotations, and other benchmarks. RESULTS: The top ranked 741 (40%) EnTDefs significantly outperform the common, naïve approach of linking distal regions to the nearest genes, and the top 10 EnTDefs perform well when applied to ChIP-seq data of other cell types. The GSE-based ranking of EnTDefs is highly concordant with ranking based on overlap with curated benchmarks of enhancer-gene interactions. Both our top general EnTDef and cell-type-specific EnTDefs significantly outperform seven independent computational and experiment-based enhancer-gene pair datasets. We show that using our top EnTDefs for GSE with either genome-wide DNA methylation or ATAC-seq data is able to better recapitulate the biological processes changed in gene expression data performed in parallel for the same experiment than our lower-ranked EnTDefs. CONCLUSIONS: Our findings illustrate the power of our approach to provide genome-wide interpretation regardless of cell type.
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Secuenciación de Inmunoprecipitación de Cromatina , Secuencias Reguladoras de Ácidos Nucleicos , ADN , Genoma Humano , Humanos , Anotación de Secuencia MolecularRESUMEN
Di(2-ethylhexyl) phthalate (DEHP) is a type of phthalate plasticizer found in a variety of consumer products and poses a public health concern due to its metabolic and endocrine disruption activities. Dysregulation of epigenetic modifications, including DNA methylation, has been shown to be an important mechanism for the pathogenic effects of prenatal exposures, including phthalates. In this study, we used an established mouse model to study the effect of perinatal DEHP exposure on the DNA methylation profile in liver (a primary target tissue of DEHP) and blood (a common surrogate tissue) of both juvenile and adult mice. Despite exposure ceasing at 3 weeks of age (PND21), we identified thousands of sex-specific differential DNA methylation events in 5-month old mice, more than identified at PND21, both in blood and liver. Only a small number of these differentially methylated cytosines (DMCs) overlapped between the time points, or between tissues (i.e. liver and blood), indicating blood may not be an appropriate surrogate tissue to estimate the effects of DEHP exposure on liver DNA methylation. We detected sex-specific DMCs common between 3-week and 5-month samples, pointing to specific DNA methylation alterations that are consistent between weanling and adult mice. In summary, this is the first study to assess the genome-wide DNA methylation profiles in liver and blood at two different aged cohorts in response to perinatal DEHP exposure. Our findings cast light on the implications of using surrogate tissue instead of target tissue in human population-based studies and identify epigenetic biomarkers for DEHP exposure.
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Exposure to particulate matter (PM) from ambient air pollution is a well-known risk factor for many lung diseases, but the mechanism(s) for this is not completely understood. Bronchial epithelial cells, which line the airway of the respiratory tract, undergo genome-wide level changes in gene expression and DNA methylation particularly when exposed to fine (<2.5 µm) PM (PM2.5). Although some of these changes have been reported in other studies, a comparison of how different concentrations and duration of exposure affect both the gene transcriptome and DNA methylome has not been done. Here, we exposed BEAS-2B, a bronchial epithelial cell line, to different concentrations of PM2.5, and compared how single or repeated doses of PM2.5 affect both the transcriptome and methylome of cells. Widespread changes in gene expression occurred after cells were exposed to a single treatment of high-concentration (30 µg/cm2) PM2.5 for 24 h. These genes were enriched in pathways regulating cytokine-cytokine interactions, Mitogen-Activated Protein Kinase (MAPK) signaling, PI3K-Akt signaling, IL6, and P53. DNA methylomic analysis showed that nearly half of the differentially expressed genes were found to also have DNA methylation changes, with just a slightly greater trend toward overall hypomethylation across the genome. Cells exposed to a lower concentration (1 µg/cm2) of PM2.5 demonstrated a comparable, but more attenuated change in gene expression compared to cells exposed to higher concentrations. There were also many genes affected by lower concentrations of PM2.5, but not higher concentrations. Additionally, repeated exposure to PM2.5 (1 µg/cm2) for seven days resulted in transcriptomic and DNA methylomic changes that were distinct from cells treated with PM2.5 for only one day. Compared to single exposure, repeated exposure to PM2.5 caused a more notable degree of hypomethylation across the genome, though certain genes and regions demonstrated increased DNA methylation. The overall increase in hypomethylation, especially with repeated exposure to PM2.5, was associated with an increase in expression of ten-eleven translocation enzymes. These data demonstrate how variations in concentration and duration of PM2.5 exposure induce distinct differences in the transcriptomic and DNA methylomic profile of bronchial epithelial cells, which may have important implications in the development of both acute and chronic lung disease.
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Early developmental environment can influence long-term health through reprogramming of the epigenome. Human environmental epigenetics studies rely on surrogate tissues, such as blood, to assess the effects of environment on disease-relevant but inaccessible target tissues. However, the extent to which environment-induced epigenetic changes are conserved between these tissues is unclear. A better understanding of this conservation is imperative for effective design and interpretation of human environmental epigenetics studies. The Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium was established by the National Institute of Environmental Health Sciences to address the utility of surrogate tissues as proxies for toxicant-induced epigenetic changes in target tissues. We and others have recently reported that perinatal exposure to lead (Pb) is associated with adverse metabolic outcomes. Here, we investigated the sex-specific effects of perinatal exposure to a human environmentally relevant level of Pb on DNA methylation in paired liver and blood samples from adult mice using enhanced reduced-representation bisulphite sequencing. Although Pb exposure ceased at 3 weeks of age, we observed thousands of sex-specific differentially methylated cytosines in the blood and liver of Pb-exposed animals at 5 months of age, including 44 genomically imprinted loci. We observed significant tissue overlap in the genes mapping to differentially methylated cytosines. A small but significant subset of Pb-altered genes exhibit basal sex differences in gene expression in the mouse liver. Collectively, these data identify potential molecular targets for Pb-induced metabolic diseases, and inform the design of more robust human environmental epigenomics studies.
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Metilación de ADN , Epigenómica , Animales , Citosina , Exposición a Riesgos Ambientales , Epigénesis Genética , Femenino , Plomo , Masculino , Ratones , EmbarazoRESUMEN
Studies in rodents and captive primates suggest that the early-life social environment affects future phenotype, potentially through alterations to DNA methylation. Little is known of these associations in wild animals. In a wild population of spotted hyenas, we test the hypothesis that maternal care during the first year of life and social connectedness during two periods of early development leads to differences in DNA methylation and fecal glucocorticoid metabolites (fGCMs) later in life. Here we report that although maternal care and social connectedness during the den-dependent life stage are not associated with fGCMs, greater social connectedness during the subadult den-independent life stage is associated with lower adult fGCMs. Additionally, more maternal care and social connectedness after den independence correspond with higher global (%CCGG) DNA methylation. We also note differential DNA methylation near 5 genes involved in inflammation, immune response, and aging that may link maternal care with stress phenotype.
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Epigénesis Genética/fisiología , Hyaenidae/psicología , Conducta Materna/fisiología , Medio Social , Estrés Psicológico/diagnóstico , Envejecimiento/genética , Envejecimiento/psicología , Animales , Metilación de ADN/fisiología , Heces/química , Femenino , Glucocorticoides/análisis , Glucocorticoides/metabolismo , Hyaenidae/genética , Hyaenidae/crecimiento & desarrollo , Masculino , Estrés Psicológico/genética , Estrés Psicológico/metabolismo , Estrés Psicológico/psicologíaRESUMEN
BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.
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Epigénesis Genética , Epigenómica/métodos , Control de Calidad , 5-Metilcitosina , Algoritmos , Islas de CpG , ADN/genética , Metilación de ADN , Epigenoma , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Sulfitos , Secuenciación Completa del Genoma/métodosRESUMEN
Phthalate plasticizers are ubiquitous chemicals linked to several cardiovascular diseases in animal models and humans. Despite this, the mechanisms by which phthalate exposures cause adverse cardiac health outcomes are unclear. In particular, whether phthalate exposures during pregnancy interfere with normal developmental programming of the cardiovascular system, and the resulting implications this may have for long-term disease risk, are unknown. Recent studies suggest that the effects of phthalates on metabolic and neurobehavioral outcomes are sex-specific. However, the influence of sex on cardiac susceptibility to phthalate exposures has not been investigated. One mechanism by which developmental exposures may influence long-term health is through altered programming of DNA methylation. In this work, we utilized an established mouse model of human-relevant perinatal exposure and enhanced reduced representation bisulfite sequencing to investigate the long-term effects of diethylhexyl phthalate (DEHP) exposure on DNA methylation in the hearts of adult male and female offspring at 5 months of age (n = 5-7 mice per sex and exposure). Perinatal DEHP exposure led to hundreds of sex-specific, differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs) in the heart. Pathway analysis of DMCs revealed enrichment for several pathways in females, including insulin signaling, regulation of histone methylation, and tyrosine phosphatase activity. In males, DMCs were enriched for glucose transport, energy generation, and developmental programs. Notably, many sex-specific genes differentially methylated with DEHP exposure in our mouse model were also differentially methylated in published data of heart tissues collected from human heart failure patients. Together, these data highlight the potential role for DNA methylation in DEHP-induced cardiac effects and emphasize the importance of sex as a biological variable in environmental health studies.
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BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, with human papillomavirus (HPV)-related HNSCC rising to concerning levels. Extensive clinical, genetic and epigenetic differences exist between HPV-associated HNSCC and HPV-negative HNSCC, which is often linked to tobacco use. However, 5-hydroxymethylation (5hmC), an oxidative derivative of DNA methylation and its heterogeneity among HNSCC subtypes, has not been studied. RESULTS: We characterized genome-wide 5hmC profiles in HNSCC by HPV status and subtype in 18 HPV(+) and 18 HPV(-) well-characterized tumors. Results showed significant genome-wide hyper-5hmC in HPV(-) tumors, with both promoter and enhancer 5hmC able to distinguish meaningful tumor subgroups. We identified specific genes whose differential expression by HPV status is driven by differential hydroxymethylation. CDKN2A (p16), used as a key biomarker for HPV status, exhibited the most extensive hyper-5hmC in HPV(+) tumors, while HPV(-) tumors showed hyper-5hmC in CDH13, TIMP2, MMP2 and other cancer-related genes. Among the previously reported two HPV(+) subtypes, IMU (stronger immune response) and KRT (more keratinization), the IMU subtype revealed hyper-5hmC and up-regulation of genes in cell migration, and hypo-5hmC with down-regulation in keratinization and cell junctions. We experimentally validated our key prediction of higher secreted and intracellular protein levels of the invasion gene MMP2 in HPV(-) oral cavity cell lines. CONCLUSION: Our results implicate 5hmC in driving differences in keratinization, cell junctions and other cancer-related processes among tumor subtypes. We conclude that 5hmC levels are critical for defining tumor characteristics and potentially used to define clinically meaningful cancer patient subgroups.
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5-Metilcitosina/análogos & derivados , Uniones Intercelulares/metabolismo , Queratinocitos/metabolismo , Papillomaviridae/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , 5-Metilcitosina/metabolismo , Movimiento Celular/genética , Metilación de ADN , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/complicaciones , Neoplasias Cutáneas/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/diagnóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/etiología , Carcinoma de Células Escamosas de Cabeza y Cuello/virologíaRESUMEN
Lead (Pb) exposure remains a major concern in the United States (US) and around the world, even following the removal of Pb from gasoline and other products. Environmental Pb exposures from aging infrastructure and housing stock are of particular concern to pregnant women, children, and other vulnerable populations. Exposures during sensitive periods of development are known to influence epigenetic modifications which are thought to be one mechanism of the Developmental Origins of Health and Disease (DOHaD) paradigm. To gain insights into early life Pb exposure-induced health risks, we leveraged neonatal dried bloodspots in a cohort of children from Michigan, US to examine associations between blood Pb levels and concomitant DNA methylation profiles (n = 96). DNA methylation analysis was conducted via the Infinium MethylationEPIC array and Pb levels were assessed via high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). While at-birth Pb exposure levels were relatively low (average 0.78 µg/dL, maximum of 5.27 ug/dL), we identified associations between DNA methylation and Pb at 33 CpG sites, with the majority (82%) exhibiting reduced methylation with increasing Pb exposure (q < 0.2). Biological pathways related to development and neurological function were enriched amongst top differentially methylated genes by p-value. In addition to increases/decreases in methylation, we also demonstrate that Pb exposure is related to increased variability in DNA methylation at 16 CpG sites. More work is needed to assess the accuracy and precision of metals assessment using bloodspots, but this study highlights the utility of this unique resource to enhance environmental epigenetics research around the world.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Plomo , Efectos Tardíos de la Exposición Prenatal , Niño , Epigenómica , Femenino , Humanos , Recién Nacido , Plomo/sangre , Plomo/toxicidad , Masculino , Michigan , Tamizaje Neonatal , EmbarazoRESUMEN
Gene set enrichment (GSE) testing enhances the biological interpretation of ChIP-seq data and other large sets of genomic regions. Our group has previously introduced two GSE methods for genomic regions: ChIP-Enrich for narrow regions and Broad-Enrich for broad regions. Here, we introduce Poly-Enrich, which has wider applicability, additional capabilities and models the number of peaks assigned to a gene using a generalized additive model with a negative binomial family to determine gene set enrichment, while adjusting for gene locus length. As opposed to ChIP-Enrich, Poly-Enrich works well even when nearly all genes have a peak, illustrated by using Poly-Enrich to characterize pathways and types of genic regions enriched with different families of repetitive elements. By comparing Poly-Enrich and ChIP-Enrich results with ENCODE ChIP-seq data, we found that the optimal test depends more on the pathway being regulated than on properties of the transcription factors. Using known transcription factor functions, we discovered clusters of related biological processes consistently better modeled with Poly-Enrich. This suggests that the regulation of certain processes may be modified by multiple binding events, better modeled by a count-based method. Our new hybrid method automatically uses the optimal method for each gene set, with correct FDR-adjustment.